ags cells bcrc 60102  (BioResource International Inc)

Journal: PLoS ONE

Article Title: H. pylori CagL-Y58/E59 Prime Higher Integrin α5β1 in Adverse pH Condition to Enhance Hypochlorhydria Vicious Cycle for Gastric Carcinogenesis

doi: 10.1371/journal.pone.0072735

Figure Lengend Snippet: ( A ) Integrin α5 and β1 expressions of AGS cells cultivated alone or co-cultivated with Hp1033 at pH 7.4, 5.4, and 4.4, respectively. Cell lysate was immunoblotted with an anti-integrin α5 and anti- integrin β1 antibody. GAPDH served as an internal control for sample normalization. The integrin α5 expression was significantly higher in trend-like fashion as the pH elevated from 4.4, 5.4, to 7.4 (*indicated p <0.05). ( B ) CagA phosphorylation of AGS cells co-cultivated with Hp1033 for 8 h at pH 7.4, 5.4 and 4.4, respectively. Cell lysate was immunoblotted with an anti-CagA, Hsp60 and anti-phospho-CagA antibody. The CagA phosphorylation at pH 5.4 was higher than that of pH 4.4 ( p <0.05). The data are shown as mean value ± standard deviation of the triplicate experiments.

Article Snippet: For assaying integrin α5β1 priming and CagA phosphorylation by H. pylori at different pH values, human gastric cancer cell lines as AGS cells (Bioresource Collection and Research Center, BCRC 60102) exposed to Hp1033 under the different pH ranges from 4.4, 5.4 to 7.4 in the culture mediums for 16 h. The cell lysates were collected to check the expression of integrin α5 or β1, and phosphorylated CagA by immunoblotting.

Techniques: Control, Expressing, Phospho-proteomics, Standard Deviation

Journal: PLoS ONE

Article Title: H. pylori CagL-Y58/E59 Prime Higher Integrin α5β1 in Adverse pH Condition to Enhance Hypochlorhydria Vicious Cycle for Gastric Carcinogenesis

doi: 10.1371/journal.pone.0072735

Figure Lengend Snippet: ( A ) Integrin α5 and β1 expressions of AGS cells cultivated alone or co-cultivated with 4 wild-type Hp1033 cagL isogenic mutants (CagL-Y58/E59 revertant, Y58D/E59, Y58/E59K, and Y58D/E59K amino acid replacement mutants) at pH 5.4 for 18 hours were analyzed by immunoblotting. There was no difference in the integrin α5 and β1 expressions among these 4 H. pylori isolates. ( B ) The active form integrin β1 of AGS cells co-cultivated with CagL amino acid replacement mutants at pH 7.4 and 5.4 were analyzed by immunoblotting. The values of the active forms of integrin β1 were normalized to total forms, and there were significant higher in CagL-Y58/E59 than in CagL-Y58D/E59K at pH 7.4, higher in CagL-Y58/E59 than other mutants at pH 5.4 ( † p <0.05), higher in CagL-Y58D/E59 than in CagL-Y58D/E59K at both pH 7.4 and pH 5.4 ( ‡ p <0.05), and higher in CagL-Y58/E59K than in CagL-Y58D/E59K at pH 7.4 and pH 5.4 ( # p <0.05). ( C ) Quartz crystal microbalance measurement of the interaction of CagL-Y58/E59 and CagL-D58/K59 with immobilized integrin α5β1. △F is the change of frequency (MHz) after different CagL proteins in range of 60 to 480 nM binding to integrin. The kd translated from △F via software (Affinity Evaluation Software v1.0, ANT technology Co., Ltd). The Kd was lower in CagL-Y58/E59 than in CagL-D58/K59, indicating the stronger binding affinity of the former. ( D ) The ability of CagA translocation was represented by CagA phosphorylation (p-CagA) levels in AGS cells co-cultivated with CagL-Y58/E59 revertant, Y58D/E59, Y58/E59K, and Y58D/E59K amino acid replacement mutants at pH 5.4 for 1 h. The value of p-CagA, normalized to CagA, was lower in the CagL-Y58D/E59K than in either CagL-Y58/E59 or CagL-Y58D/E59, (* p <0.05). The data were mean ± standard deviations of the triplicate experiments. ( E ) There were significant differences of IL-8 levels triggered between CagL-Y58/E59 vs . CagL-Y58D/E59, CagL-Y58/E59 vs . CagL-Y58D/E59K, CagL-Y58D/E59 vs . CagL-Y58D/E59K, and CagL-Y58/E59K vs . CagL-Y58D/E59K isolates ( p <0.05).

Article Snippet: For assaying integrin α5β1 priming and CagA phosphorylation by H. pylori at different pH values, human gastric cancer cell lines as AGS cells (Bioresource Collection and Research Center, BCRC 60102) exposed to Hp1033 under the different pH ranges from 4.4, 5.4 to 7.4 in the culture mediums for 16 h. The cell lysates were collected to check the expression of integrin α5 or β1, and phosphorylated CagA by immunoblotting.

Techniques: Western Blot, Binding Assay, Software, Translocation Assay, Phospho-proteomics

Journal: PLoS ONE

Article Title: H. pylori CagL-Y58/E59 Prime Higher Integrin α5β1 in Adverse pH Condition to Enhance Hypochlorhydria Vicious Cycle for Gastric Carcinogenesis

doi: 10.1371/journal.pone.0072735

Figure Lengend Snippet: The AGS cells were co-cultivated with CagL-Y58/E59 or CagL-Y58D/E59K H. pylori at pH 7.4 or pH 5.4 to check the expression ratios of ( A ) phospho-FAK & FAK, ( B ) phospho-EGFR & EGFR, and ( C ) phospho-AKT & AKT. There were significant differences in phosphorylation level of FAK, AKT at pH 5.4 and EGFR at pH 5.4 and 7.4 triggered between CagL-Y58/E59 or CagL-Y58D/E59K H. pylori (* p <0.05). The data were mean ± standard deviations. Each experiment was repeated in triplicate.

Article Snippet: For assaying integrin α5β1 priming and CagA phosphorylation by H. pylori at different pH values, human gastric cancer cell lines as AGS cells (Bioresource Collection and Research Center, BCRC 60102) exposed to Hp1033 under the different pH ranges from 4.4, 5.4 to 7.4 in the culture mediums for 16 h. The cell lysates were collected to check the expression of integrin α5 or β1, and phosphorylated CagA by immunoblotting.

Techniques: Expressing, Phospho-proteomics